The present invention relates to a DNA involved in the function of controlling autonomous replication in a bacterial cell belonging to the genus Gluconobactor, a plasmid vector containing said DNA, a transformant transformed with said plasmid vector and a method for producing a physiologically active substance such as an enzyme, comprising culturing said transformant.
The plasmids belonging to the genus Gluconobactor are known to include pF2, pF3, pF4, shuttle vector pFG5B (combined plasmid of pHSG298 (plasmid of Escherichia coli) and pF2), pFG14A (combined plasmid of pHSG298 and pF3), pFG15A (combined plasmid of pHSG298 and pF4) (all of which disclosed in WO95/23220) and the like. With regard to these plasmids, however, the specific position of the plasmid that controls autonomous replication in the bacterial cell belonging to the genus Gluconobactor or the sequence thereof has not been known at all.
An object of the present invention is to specify the region of the nucleotide sequence of plasmid pF4 derived from the genus Gluconobactor (Gluconobactor oxydans T-100; see W095/23220), which is necessary for replication in the bacterium belonging to the genus Gluconobactor. Another object of the present invention is to provide a plasmid vector containing a region essential for the replication but is free of the region not essential for the replication, or compact pF4, and a transformant transformed with this plasmid vector. A further object of the present invention is to provide a plasmid vector containing, besides the above-mentioned region, a structural gene capable of expression in a host cell (hereinafter this plasmid is also referred to as recombinant expression vector), a transformant transformed with this recombinant expression vector and a method for producing a physiologically active substance, which comprises culturing this transformant and harvesting a physiologically active substance, such as enzyme and the like, which has been expressed in the culture.
The present inventors have conducted intensive studies in an attempt to solve the above-mentioned problems, and succeeded in specifying the region of the nucleotide sequence of plasmid pF4 derived from the genus Gluconobactor, which is essential for the replication in bacterial cells belonging to the genus Gluconobactor. They have also succeeded in shortening plasmid pF4 by leaving the region essential for the replication but removing a part or the whole of the region not essential for the replication, as well as in preparing a vector containing the shortened plasmid and a recombinant expression vector additionally containing a structural gene, which resulted in the completion of the present invention.
Accordingly, the present invention provides the following.
(1) A DNA derived from plasmid pF4, which contains a region involved in the control of autonomous replication in a bacterial cell belonging to the genus Gluconobactor, with or without a part of the polynucleotide region not essential for the autonomous replication therein, except pF4 itself.
(2) The DNA of the above (1), substantially comprising, as the region involved in the control of autonomous replication in the bacterial cell belonging to the genus Gluconobactor, a part or the entirety of the polynucleotide of nucleotide Nos. 2897-3969 region of Sequence Listing SEQ:ID No. 1, or substantially comprising a part or the entirety of polynucleotide of the nucleotide Nos. 2897-3969 region of Sequence Listing SEQ:ID No. 1, wherein at least one nucleotide has been deleted, substituted, added or inserted.
(3) A DNA substantially having a nucleotide sequence depicted in Sequence Listing SEQ:ID No. 1 or a DNA having the same nucleotide sequence depicted in Sequence Listing SEQ:ID No. 1 except that at least one nucleotide has been deleted, substituted, added or inserted.
(4) A plasmid vector comprising the DNA of the above (1) or (2).
(5) The plasmid vector of the above (4), comprising a DNA involved in autonomous replication in a bacterial cell belonging to the genus Gluconobactor and a DNA involved in autonomous replication in a host other than the bacterial cell belonging to the genus Gluconobactor, particularly Escherichia coli. 
(6) The plasmid vector of the above (4) or (5), comprising, within the plasmid vector, a structural gene capable of expression in a host.
(7) The plasmid vector of the above (6), wherein the structural gene capable of expression in the host encodes a physiologically active substance.
(8) The plasmid vector of the above (7), wherein the physiologically active substance is L-sorbose dehydrogenase and/or L-sorbosone dehydrogenase.
(9) A transformant transformed with the plasmid vector of any of the above (4)-(8).
(10) A method for producing a physiologically active substance, comprising culturing the transformant transformed with the plasmid vector of the above (7) to express the physiologically active substance, and harvesting the physiologically active substance.
(11) A method for producing L-sorbose dehydrogenase and/or L-sorbosone dehydrogenase, comprising culturing a transformant transformed with the plasmid vector of the above (8) to express L-sorbose dehydrogenase and/or L-sorbosone dehydrogenase, and harvesting the L-sorbose dehydrogenase and/or L-sorbosone dehydrogenase.
(12) A method for producing 2-keto-L-gulonic acid, comprising culturing a transformant transformed with the plasmid vector of the above (8) to express L-sorbose dehydrogenase and L-sorbosone dehydrogenase.